Phospholipase A2s (PLA2s) are a ubiquitous family of enzymes which have been shown to mediate several cellular responses including metabolism, phospholipid digestion, host defense, apoptosis and signal transduction. The PLA2 enzymes are abundant in nature with a strong presence in snake and insect venoms and the pancreatic fluids of mammals (Kramer and Sharp, FEBS Lett., 1997, 410, 49-53).
Although there are several forms of PLA2 enzymes in mammalian tissues, each with a distinct localization, structure, regulation and function, the common role of PLA2 enzymes is to maintain the integrity of the cell membrane. Following activation, PLA2 enzymes hydrolyze phospholipids, producing free fatty acids and lysophospholipids. These lipid hydrolysis products have been shown to act as second messengers and metabolic precursors for molecules having major implications in inflammation and degenerative disorders such as Alzheimer's disease (Murakami et al., Crit. Rev. Immunol., 1997, 17, 225-283). Consequently, these enzymes have been the topic of intense study in an effort to develop novel therapeutic agents against inflammatory and degenerative diseases.
Phospholipase A2 Group IV (also known as cytosolic Phospholipase A2), a member of the PLA2 family of enzymes, is found in most mammalian tissues and preferentially cleaves arachidonate-containing phospholipids. In mammalian cells, the predominant fatty acid component of phospholipids is arachidonic acid. This fatty acid once liberated from the phospholipid is the precursor to a group of pro-inflammatory molecules known as eicosanoids which include prostaglandins, leukotrienes, and thromboxanes (Kramer and Sharp, FEBS Lett., 1997, 410, 49-53).
Phospholipase A2 Group IV is unique in that it is the only member of the PLA2 family that exhibits properties of a receptor-regulated protein. This means that the activation of Phospholipase A2 Group IV is triggered in response to extracellular stimuli including ligand binding. A variety of stimuli have been shown to activate Phospholipase A2 Group IV including growth factors, cytokines, mitogens, vasoactive peptides, integrin engagement, and interferons (Kramer and Sharp, FEBS Lett., 1997, 410, 49-53). In addition, stress stimuli such as oxidation, hyperglycemia and UV light have also been shown to activate Phospholipase A2 Group IV (Murakami et al., Crit. Rev. Immunol., 1997, 17, 225-283).
Manifestations of altered Phospholipase A2 Group IV regulation appear in both cell and tissue injury as well as disease states and the products of Phospholipase A2 Group IV hydrolysis have been shown to contribute to inflammatory and degenerative diseases. It is known that during ischemia, the concentration of free fatty acids in the brain increases, the most predominant being arachidonic acid presumably liberated by the action of Phospholipase A2 Group IV (Kramer et al., J. Lipid Mediat. Cell Signal., 1996, 14, 3-7). Using antibodies to Phospholipase A2 Group IV, other studies have shown that there is an elevated level of the Phospholipase A2 Group IV protein in the brains of Alzheimer's disease patients suggesting that an active inflammatory process occurs in the disorder (Stephenson et al., Neurobiol. Dis., 1996, 3, 51-63).
Furthermore, the phosphorylation state of Phospholipase 10 A2 Group IV has been implicated in the process of apoptosis. It is currently believed that sustained phosphorylation is necessary for the activation of Phospholipase A2 Group IV in apoptotic cells (OBrien et al., J. Immunol., 1998, 161, 1525-1532).
Inhibitors of Phospholipase A2 Group IV have been used to elucidate the role of Phospholipase A2 Group IV in hypotonic cell swelling in human neuroblastoma cell lines. These studies showed that AACOCF3 (arachidonyl trifluoromethylketone) reduced the release of arachidonic acid from the cells by inhibiting Phospholipase A2 Group IV thereby reducing cellular swelling (Basavappa et al., J. Neurophysiol., 1998, 79, 1441-1449). Another inhibitor, 3,3-Dimethyl-6-(3-lauroylureido)-7-oxo-4-thia-1-azabicyclo[3,2,0] heptane-2-carboxylic acid, a beta lactam, was shown to partition into the phospholipid bilayer and compete with the phospholipid substrate for the active site of Phospholipase A2 Group IV (Burke et al., J. Enzyme Inhib., 1998, 13, 195-206).
Wu et al. demonstrated that an antisense oligonucleotide targeted to the initiation site of human Phospholipase A2 Group IV and the Phospholipase A2 Group IV inhibitor, 4-bromophenacyl bromide significantly inhibited TNF-induced cytotoxicity in U937 human leukemic cells and concluded that disruption of Phospholipase A2 Group IV activation may represent a possible mechanism by which leukemia cells may become resistent to TNF-mediated apoptosis (Wu et al., Cancer Res., 1998, 58, 633-640). Other studies using an antisense targeted to the first 21 bases in the coding sequence of the high molecular weight Phospholipase A2 Group IV showed that increased Phospholipase A2 Group IV synthesis occurs when skin is exposed to UV doses sufficient to cause erythema and that an S-oligonucleotide antisense to Phospholipase A2 Group IV and methyl arachidonate fluorophosphate, a Phospholipase A2 Group IV specific inhibitor, blocked this effect (Gresham et al., Am. J. Physiol., 1996, 270, C1037-1050). In addition, monocytes cultured in the presence of an antisense oligonucleotide targeted to 16 bases just downstream from the initiation site of Phospholipase A2 Group IV showed reduced chemokine-induced chemotaxis (Locati et al., J. Biol. Chem., 1996, 271, 6010-6016).
These strategies, however, have not been expanded to the therapeutic level of treatment and consequently there remains a long felt need for additional agents capable of effectively inhibiting Phospholipase A2 Group IV function.